2.4 LAMP and conventional PCR
WarmStart® LAMP Kit (New England Biolabs, Ipswich, MA) used for LAMP assay. General protocol of LAMP was flowed by manufacture’s guideline in a 25 μl reaction mixture. For the conventional PCR, TOYOBO KOD‐FX TaqTM (Toyobo Life Science, Osaka, Japan) used in this study. Species-specific primers with the following PCR amplification protocol: a 2‐min denaturing step at 94°C and a PCR amplification cycle consisting of denaturing at 94°C for 20 s, annealing at 60°C for 20 s, and extension at 68°C for 30 s, which was repeated 35 times. The amplified DNA fragments were separated using 1.5% agarose gel electrophoresis, visualized with SYBR green (Life Technologies, Grand Island, NY). Used insect samples were collected in Korean cornfields except S. littoralis . MPI-CE lab strain of S. littoralisand mitochondrial genome sequenced S. frugiperda samples (Jeju, Muan and Miryang) used. Pheromone traps were used as previously reported to collect samples (S. exigua, S. litura, Mythimna separate, andHelicoverpa armigera ). Traps were set in Pyeongchang (37°40.53 N, 128°43.49 E), Hongchen (37°43.35 N, 128°24.33 E) and Gangneung (37°36.56 N, 128°45.59 E)(J. Kim, Kwon, Park, & Maharjanm, 2018). DNA samples were prepared using DNAzol from trapped adults. Two or three biological DNA samples were used in each LAMP and PCR.
RESULTS